Self-renewing epithelial tissues in skin, cornea, intestine and prostate have been shown to contain stem cells that provide the tissue's regenerative potential. Surprisingly, stem cells have not been definitively identified in the bladder. The goal of this application is to identify, characterize and enrich adult urothelial stem cells. Identifying cell surface proteins that are over- or under-expressed in stem cells relative to differentiated cells would provide a battery of positive or negative stem cell markers, respectively, to facilitate their isolation. Stem cells also have unique transport proteins and conserved enzymes that clear the cell of toxins. Flow cytometry and cell sorting on the basis of the expression of these transport proteins has been used to isolate stem cells from other tissues. We will assess the feasibility of applying this strategy to enriching for urothelial stem cells. Using known markers for stem cells in other systems, urothelium will be sorted into stem and non-stem cell populations. These cell populations will be compared in vitro for colony forming efficiency, proliferation, long-term growth and ability to regenerate bladder tissue in bioengineered tissue constructs. The pluripotency of stem cells will also be assessed by combining the sorted urothelial populations with non-urologic mesenchymal cells from another species and growing the xenografts under the renal capsule of a nude mouse. Bioengineering and gene therapy are currently being investigated and utilized for the treatment of urologic diseases in humans. The identification, characterization and enrichment of adult urothelial stem cells will have significant impact on laboratory and clinical investigation of normal and neoplastic urothelial differentiation, bioengineering, gene therapy and cancer treatment. Successful bioengineering of durable urologic organs may depend upon the harvest and transplantation of stem cells. Successful and efficient gene therapy depends upon the transduction of stem cells that will propagate the intended gene and/or gene product. A better understanding and availability of urothelial stem cells will also allow further investigation of normal and abnormal differentiation pathways. This will lead to further study of cancer stem cells and better treatments for transitional cell carcinoma. PUBLIC HEALTH RELEVANCE: Stem cells carry a tissue's regenerative potential for the life of the organism. Durable and successful bioengineering, gene therapy and cancer treatment depend upon the existence of stem cells. The identification, characterization and enrichment of urinary bladder stem cells will lead to better treatment of urologic diseases.